This research project includes an investigation of the activation mechanism of the classical complement pathway initiator, C1. Additionally, clinically useful methods will be developed for the quantitation of native C1 and free C1 subunits, and for the detection and quantitation of complement activation in human serum. Finally, physiological mechanisms of complement control will be investigated. The mechanism of activation of human C1 will be investigated. Physicochemical and functional differences of precursor and activated C1q will be studied by techniques including differential sedimentation, circular dichroism spectroscopy, SDS polyacrylamide gel electrophoresis, isoelectric focussing, and hemolytic titration. Direct evidence for conformational changes in the C1 macromolecule with activation will be sought. In addition to some of the physical methods mentioned above, a differential radiolabeling technique will be developed as a probe for conformational changes. Kinetic studies and nuclear magnetic resonance spectroscopy will be used to investigate the importance of calcium redistribution or release during C1 activation. The high affinity lanthanide cations will also be used in these studies. The fate of the C1 molecule after activation will be followed. Immunochemical techniques utilizing double diffusion and single radial diffusion will be developed to detect and quantitate macromolecular C1 and free C1 subunits in normal and pathological sera. Furthermore methodology for detecting C1 activation in human serum will be developed. Three approaches will be used: 1) Advantage will be taken of the difference in the Ouchterlony patterns of C1 and C1'. 2) Antisera will be produced to neoantigens formed in C1 during activation. 3) A radioimmunoassay based on the binding of activated C1' to 125I-C1 inactivator will be developed. The sera from patients with different immunological diseases will be tested by these techniques. Finally, the mechanisms of control of C1 activation in human serum will be investigated. The role of C1' inactivator as well as any other possible serum regulators will be studied.